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Electronic Supplementary MaterialJournal: Psychopharmacology
Electronic Supplementary Material
Journal: Psychopharmacology
In-vivo and In-vitro attenuation of naloxone precipitated experimental opioid withdrawal
syndrome by insulin and selective KATP channel modulator
Prabhat Singh a, Bhupesh Sharma b, c, *, Surbhi Gupta d and B. M. Sharma e
Neuropharmacology Lab, Department of Pharmacology, School of Pharmacy, BIT, India
Conscience Research, Delhi, India
a Research Student, Neuropharmacology Lab., Department of Pharmacology, School of
Pharmacy, Bharat Institute of Technology, Partapur Bypass, Meerut, Pin- 250103, Uttar Pradesh,
India. +91-954-8116443; sharmaslab2@gmail.com
b Associate Professor and Head, Department of Pharmacology, School of Pharmacy, Bharat
Institute of Technology, Partapur Bypass, Meerut, Pin- 250103, Uttar Pradesh, India
+91-995-8219190, +91-879-1636281; drbhupeshresearch@gmail.com
c Chief Consultant, CNS Pharmacology, Conscience Research, Pocket F-233, B, Dilshad Garden,
Delhi-110095 India. +91-995-8219190; conscienceresearch@scientist.com
d Research Student, Neuropharmacology Lab., Department of Pharmacology, School of
Pharmacy, Bharat Institute of Technology, Partapur Bypass, Meerut, Pin- 250103, Uttar Pradesh,
India. +91-992-7932746; sharmaslab3@gmail.com
e Assistant Professor, Department of Pharmacology, School of Pharmacy, Bharat Institute of
Technology, Partapur Bypass, Meerut, Uttar Pradesh, India
* Corresponding Author: Dr. Bhupesh Sharma
Category of paper: Original Investigation
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Electronic Supplementary MaterialJournal: Psychopharmacology
Material and Method
Animals
Swiss albino mice (25±2g) and Wistar rats (250±20g), of either sex, were maintained on
a standard laboratory diet and having free access to tap water. To control for sex-related
variations in the results, male and female mice were divided equally among all treatment groups.
Animals were housed in the departmental animal house and were exposed to 12 hour light and 12
hour dark cycle. The Experiments were conducted in a semi sound-proof laboratory. The
experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC),
whereas the animals were taken care by following the guidelines of the Committee for the
Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of
Environment and Forests, Government of India (Reg. No. 25/230/2011/AWD/CPCSEA). Mice
were used for in-vivo studies and Wistar rats (ileum) were used for in-vitro studies.
Drugs and chemicals
Morphine sulphate and naloxone were obtained from Troikaa pharmaceuticals Ltd.,
India. Gliclazide was obtained from Morepen laboratories Ltd., India. Insulin (Huminsulin,
biphasic isophane insulin injection IP) was obtained from Gland Pharma Ltd., India. 5,5`-
dithiobis(2-nitrobenzoic acid) (DTNB) and N-naphthylethylenediamine were purchased from
Sigma Aldrich, USA.
Morphine, naloxone and insulin were dissolved in 0.9% saline solution, prepared in
triple distilled water and gliclazide was suspended in 0.5% carboxy methyl cellulose (CMC). All
drug solutions were freshly prepared before use. Selection of doses and the dosing schedule were
based on previously published reports from other labs.
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Electronic Supplementary MaterialJournal: Psychopharmacology
For in-vivo study, morphine (5mg/kg; intraperitoneally), naloxone (8mg/kg;
intraperitoneally), insulin (4 and 8 I.U/kg/day; intraperitoneally) and gliclazide (3 and
6mg/kg/day; orally) were administered to mice (Jafari et al. 2004; Satyanarayana and Kilari
2006; Rehni and Singh 2012). For in-vitro study the concentration of morphine (10−5 M),
naloxone (10−5 M), gliclazide (5×10-6 M and 5×10-5 M) and insulin (1x10-6 M and 1x10-5 M) were
used (Thomson et al. 1997; Rehni and Singh 2012; Sliwinska et al. 2012). The duration of action
of insulin and gliclazide are 18-24 hours and 12-18 hours, respectively, so the treatment drugs
were administered once daily for 6 days after 1 hour of morphine injection.
Induction of morphine withdrawal syndrome (In-vivo)
To develop dependence, morphine (5 mg/kg, intraperitoneally) was administered twice
daily (at 08: 00 and 20: 00 h) for 6 days. However, the test drug/vehicle was administered daily
(at 10:00 am) after one hour injection of morphine (Rehni and Singh 2012). The withdrawal
signs were precipitated by injecting naloxone (8 mg/kg, intraperitoneally), 2 hours after the final
injection of morphine on the 6th day. This in-vivo model has been extensively used for induction
of morphine withdrawal syndrome in various laboratories (Way et al. 1969; Rehni and Singh
2012). Immediately after a naloxone injection, mice were individually placed in an observation
chamber and behavioural observations were made in initial 30 min. The observations were
carried out in a transparent Perspex observation chamber with dimensions of 30cm×30cm×30
cm. Two observers, which were blind to the treatment schedule, simultaneously observed each
animal for all of the withdrawal measures and the mean value of both observations were
recorded. The withdrawal symptoms are reported as a summary of all of the signs that were seen.
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Induction of experimental morphine withdrawal response in isolated rat ileum (In-vitro)
Morphine withdrawal by naloxone can be induced in-vitro (rat ileum), which has been
utilized previously for screening of agents as well as for assessing morphine withdrawal
syndrome. Naloxone contracture (basal and treatment) was measured in terms of tension ratio,
using isolated rat ileum preparation on student organ bath assembly. Briefly, overnight fasted
adult rats (250±20 g), were killed by injecting high doses of thiopental sodium followed by
carotid bleeding. A small 2-3 cm section of ileum was isolated from the intestine. Rat ileum
preparation was prepared by tying a loop on one end of the tissue and tying another thread on a
diagonally opposite aspect in order to ensure the complete opening up of the linen of the tissue
while the same was mounted on the tissue bath. The tissue was then suspended in an organ bath
containing 20 ml of Krebs solution (NaCl 118, KCl 4.75, K2HPO4 1.2, CaCl2 1.26, MgSO4 1.2,
NaHCO3 25 and glucose 11.1 mM) at 37°C, aerated with 95% O2 and 5% CO2. A resting tension
of 1 g was applied to the tissue. The tissue was allowed to equilibrate for 40-60 min, and three
responses to acetylcholine (ACh, 10−6 M) were obtained so that the withdrawal response could be
expressed as the percentage of a particular mean ACh response. The experimental procedure was
similar to that described for guinea pig ileum previously (Valeri et al. 1995; Capasso and
Sorrentino 1997; Rehni and Singh 2012). Morphine (10−5 M) was added to the bath and the tissue
was exposed to the opioid agonist for a period of 4 min. Naloxone (10−5 M) was then added in
the bath to elicit a strong opioid withdrawal contracture in the morphine withdrawn rat ileum
(Rehni and Singh 2012). After a washout, another ACh response was obtained (to verify whether
the ileum’s response was modified after withdrawal contracture). After a 10 min resting period,
test drug/vehicle (varying concentrations as per the protocol) was added in the bath along with a
4 min exposure of the ileum then added to elicit a response. Following a washout, ACh response
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was repeated to affirm the functional ability of the tissue. Moreover, in order to avoid the
development of tolerance to repeated morphine exposures, each preparation was exposed to three
challenges with morphine and naloxone. Naloxone per se did not produce any effect on naive
preparations or those washed after morphine contact.
Naloxone contracture- The size of the contracture produced by naloxone challenge in all the
groups was expressed as a fraction of the mean contraction obtained with ACh in the isolated
morphine treated rat ileum preparation, calculated in terms of tension ratio as follows:
Tension Ratio = (Response to naloxone/Mean acetylcholine response) × 100
Assessment of withdrawal syndrome and overall morphine withdrawal severity (OMWS)
Naloxone, an opioid receptor antagonist, is commonly used for induction of withdrawal
syndrome (WS) in morphine withdrawal animals. Various behavioural parameters such as: Fore
paw tremors (FPT), wet dog shake (WDS), straightening, sneezing, jumping frequency (JF),
rearing frequency (RF), fore paw licking (FPL) and circling behaviour (CB) in mice were
assessed to evaluate the overall severity of WS in all the groups.
Animals were observed for each of the behavioural parameters for a period of 30 min.
Forepaw tremors (shakes of paws unrelated to grooming), wet dog shake (full body shakes
unrelated to grooming or lateral rotatory movements of the trunk to shake excessive water
present on the body hair away), straightening [the state of animal when, during locomotion, the
anterior aspect of the body (supported by forepaws) pulls the remaining body forward while its
hind paws remain stationary] and sneezing (an involuntary reflex behaviors that forcibly expels
air out of the respiratory tract) (Rabbani et al. 2012; Enquist et al. 2012), jumping frequency (A
spring or leap off the surface by a muscular effort of the legs and feet or escape attempts, all four
paws off the floor were defined as a jumping response) (Rehni and Singh 2012; Enquist et al.
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2012), frequency of rearing (repetitive raising of the frontal aspect of the mouse body while
balancing the body on the hind paws), fore paw licking (the mouse licking both of its forepaws at
least once) and circling (completions of a complete circle of the entire observation chamber were
recorded as a unit of circling behavior) were measured that were used to quantify the WS in mice
(Rehni and Singh 2012). Further, an overall morphine withdrawal severity was also measured in
all the groups, where mean frequencies of all the parameters of respective groups were summed
up. This suggests that overall morphine withdrawal severity is in proportional to every
withdrawal parameters.
Overall morphine withdrawal severity was calculated as:
OMWS = JF + RF+ FPT + CB + WDS + straightening + sneezing + FPL
Whereas, OMWS– Overall morphine withdrawal severity; JF– mean jumping frequencies; RF–
mean frequencies of rearing; FPT– mean frequencies of fore paw tremors; CB– mean
frequencies of circling behaviour; WDS– mean frequencies of wet dog shake; Sneezing– mean
sneezing frequencies; FPL– mean frequencies of fore paw licking
Biochemical assessment
Dissection and homogenization
Immediately after the estimation of behavioural parameters, the animals were sacrificed
by decapitation. Brain of each animal was separated by putting on ice and weighed individually.
10% (w/v) tissue homogenate was prepared in 0.1 M phosphate buffer (pH 7.4). The homogenate
was centrifuged at 10,000 g at 4°C for 15 min. An aliquot of supernatant was separated and used
for biochemical estimations.
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Assessment of serum glucose level
Morphine administration or its removal has been found to induce alterations in glucose
levels (Simpkins et al. 1990). Due to this reason the blood sample was collected by retro-orbital
bleeding for estimation of serum glucose. The blood was kept at room temperature for 30 min
and then centrifuged at 4000 rpm for 15 min to separate serum which was then used for
estimation of serum glucose level. The fasting blood glucose level was estimated
spectrophotometerically (UV-1800 ENG 240V; Shimadzu Coorporation, Japan) at 550 nm by
glucose per-oxidase (GOD-POD) method using commercially available kit (Sharma and Singh
2010).
Assessment of brain malondialdehyde (MDA) levels
Morphine dependent animals have been shown to increase the levels of malondialdehyde
(MDA) in the brain (Abdel-Zaher et al. 2011). Brain thiobarbituric acid reactive substances
(TBARS), was measured spectrophotometerically (UV-1800 ENG 240V; Shimadzu
Coorporation, Japan) at 532 nm (Sharma and Singh 2008a; Sharma and Singh 2010; Sharma and
Singh 2012c). The quantitative measurement of TBARS, an index of lipid peroxidation in brain
was performed. 0.2 ml of supernatant of the homogenate was pipetted out in a test tube, followed
by the addition of 0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 30% acetic acid (pH 3.5),
1.5 ml of 0.8% of thiobarbituric acid and the volume was made up to 4 ml with distilled water.
The test tubes were incubated for 1 h at 950C, then cooled and added 1 ml of distilled water,
followed by the addition of 5 ml of n-butanol-pyridine mixture (15:1 v/v). The tubes were
centrifuged at 4000 g for 10 min. The absorbance of developing pink colour was measured
spectrophotometerically at 532 nm. A standard calibration curve was prepared using 1-10 nM of
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1,1,3,3-tetra methoxy propane. The TBARS value was expressed as nanomoles per gram wet
weight of the brain (Sharma and Singh 2008b).
Assessment of brain glutathione (GSH) level
It has been reported that naloxone in morphine dependent animals, reduces the levels of
glutathione in the brain (Abdel-Zaher et al. 2011). Because of this the different glutathione
content in the brain was estimated according to the different methods such as reduced
glutathione. The reduced GSH content in the brain was estimated spectrophotometerically (UV-
1800 ENG 240V; Shimadzu Coorporation, Japan) at 412 nm. Briefly, the supernatant of the
homogenate was mixed with trichloroacetic acid (10% w/v) in 1:1 ratio. The tubes were
centrifuged at 1000 g for 10 min at 40C. The supernatant obtained (0.5 ml) was mixed with 2 ml
of 0.3 M disodium hydrogen phosphate. Then 0.25 ml of 0.001 M freshly prepared DTNB [5,5`-
dithiobis (2-nitro benzoic acid) dissolved in 1% w/v sodium citrate] was added and absorbance
was noted spectrophotometerically at 412 nm. A standard curve was plotted using 10-100 μM of
the reduced form of glutathione and results were expressed as micromoles of reduced glutathione
per gram wet weight of the brain (Sharma and Singh 2008a; Sharma and Singh 2013).
Assessment of brain nitrite/nitrate level
Naloxone in morphine dependent animals has been reported to increase the levels of
nitrite/nitrate in the brain (Abdel-Zaher et al. 2011). Brain nitrite concentration was measured
spectrophotometerically (UV-1800 ENG 240V; Shimadzu Coorporation, Japan) at 545 nm
(Sharma and Singh 2011a; Sharma and Singh 2012a; Sharma and Singh 2013). Briefly, 400 μl of
carbonate buffer (pH 9.0) was added to 100 μl of brain or standard sample followed by the
addition of small amounts (0.15 g) of copper-cadmium alloy. The tubes were incubated at room
temperature for 1 hour to reduce nitrate to nitrite. The reaction was stopped by adding 100 μl of
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0.35 M sodium hydroxide. Following this, 400 μl of zinc sulphate solution (120 mM) was added
to deproteinate the brain samples. The samples were allowed to stand for 10 min and then
centrifuged at 4000 g for 10 min. Greiss reagent (250 μl of 1.0% sulphanilamide prepared in 3 N
HCl and 250 μl of 0.1% N-naphthylethylenediamine (prepared with water) was added to aliquots
(500 μl) of clear supernatant and brain nitrite was measured spectrophotometerically at 545 nm.
The standard curve of sodium nitrite (5 to 50 μM) was plotted to calculate the concentration of
serum nitrite (Sharma and Singh 2011b; Sharma and Singh 2012b).
Assessment of brain calcium (Ca+2) levels
The administration of opiates modifies the content and movement of Ca+2 in the brain.
Morphine dependence is associated with a large increase in the content of Ca+2 in the brain of
mice (Bongianni et al. 1986). The increased Ca+2 levels in the brain were measured by using an
Auto analyzer (RMSBCA 201). The test procedure was performed according to method of Carl
et al. (1996).
Experimental protocol
In the present investigation, for in-vivo studies total twelve groups were employed with
each group comprising of eight Swiss albino mice of either sex and for in-vitro studies eight
groups were employed, with each group comprising of six Wistar rats of either sex (Figure 1).
Protocol for morphine withdrawal syndrome in mice (In-vivo):
Group I and II-- Vehicle control group (0.9% w/v saline and 0.5% CMC): Animals were
administered with saline (10 ml/kg/day; intraperitoneally) and CMC (10 ml/kg/day, orally) for 6
days respectively.
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Group III, IV, V and VI-- Insulin (dose 1 and dose 2) and gliclazide (dose 1 and dose 2) per
se: Animals were administered with insulin (4 and 8 I.U/kg/day; intraperitoneally) and gliclazide
(3 and 6 mg/kg/day; orally) for 6 days.
Group VII-- Morphine treatment group: Animals were administered with morphine (5
mg/kg/day; intraperitoneally) for 6 days.
Group VIII-- Morphine + Naloxone treatment group: All animals were administered with
morphine (5 mg/kg/day; intraperitoneally) for 6 days. On 6th day these animals were
administered with naloxone (8 mg/kg; intraperitoneally) after 2 hours of morphine treatment.
Group IX-- Morphine + Insulin (dose 1) + Naloxone treatment group: All animals were
administered with morphine (5 mg/kg/day; intraperitoneally) and simultaneously 1 hour later
with insulin (4 I.U/kg/day; intraperitoneally) for a period of 6 days. On the 6th day, naloxone (8
mg/kg, intraperitoneally) was administered to the animals after 2 hours of morphine treatment.
Group X-- Morphine + Insulin (dose 2) + Naloxone treatment group: All animals were
administered with morphine (5 mg/kg/day; intraperitoneally) and simultaneously 1 hour later
with insulin (8 I.U/kg/day; intraperitoneally) for a period of 6 days. On the 6th day, naloxone (8
mg/kg; intraperitoneally) was administered to the animals after 2 hours of morphine treatment.
Group XI-- Morphine + Gliclazide (dose 1) + Naloxone treatment group: All animals were
administered with morphine (5mg/kg/day; intraperitoneally and simultaneously 1 hour later with
gliclazide (3 mg/kg/day; orally) for a period of 6 days. On the 6th day, naloxone (8 mg/kg;
intraperitoneally) was administered to the animals after 2 hours of morphine treatment.
Group XII-- Morphine + Gliclazide (dose 2) + Naloxone treatment group: All animals were
administered with morphine (5 mg/kg/day; intraperitoneally and simultaneously 1 hour later with
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gliclazide (6 mg/kg/day; orally) for a period of 6 days. On the 6th day, naloxone (8 mg/kg;
intraperitoneally) was administered to the animals after 2 hours of morphine treatment.
Protocol for morphine withdrawal response in isolated rat ileum preparation (In-vitro):
Group I-- Morphine treatment: Basal contraction was obtained in the morphine dependent rat
ileum.
Group II--Insulin treatment (10x10-6 M): Basal contraction was obtained in insulin (dissolved
in 0.9% saline) treated rat ileum. The concentrations of insulin achieved in the bath employed to
assess their effect on withdrawal response was 10x10-6 M.
Group III--Gliclazide treatment (50x10-6 M): Basal contraction was obtained in gliclazide
(dissolved in 0.9% saline) treated rat ileum. The concentrations of insulin achieved in the bath
employed to assess their effect on withdrawal response was 50×10-6 M.
Group IV--Morphine-naloxone: Basal naloxone contraction was obtained in the morphine
dependent rat ileum. After 10 min resting periods, the same tissue was subjected to the vehicle
(0.9% saline) for insulin and gliclazide along with morphine treatment and the effect of naloxone
on such a tissue was then tested.
Group V--Insulin treatment (1x10-6 M) + Morphine-naloxone response: Basal naloxone
contraction was obtained in the morphine dependent rat ileum. After 10 min resting periods, the
same tissue was subjected to insulin treatment (dissolved in 0.9% saline) along with morphine
treatment and the effect of naloxone on such a tissue was then tested. The concentrations of
insulin achieved in the bath employed to assess their effect on withdrawal response was 1x10-6 M
for group V.
Group VI--Insulin treatment (10x10-6 M) + Morphine-naloxone response: Basal naloxone
contraction was obtained in the morphine dependent rat ileum. After 10 min resting periods, the
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same tissue was subjected to insulin treatment (dissolved in 0.9% saline) along with morphine
treatment and the effect of naloxone on such a tissue was then tested. The concentrations of
insulin achieved in the bath employed to assess their effect on withdrawal response was 10x10-6
M for group VI.
Group VII--Gliclazide treatment (5×10-6 M) + Morphine-naloxone response: Basal naloxone
contraction was obtained in the morphine dependent rat ileum. After 10 min resting periods, the
same tissue was subjected to gliclazide treatment (dissolved in 0.9% saline) along with morphine
treatment and the effect of naloxone on such a tissue was then tested. The concentrations of
gliclazide achieved in the bath employed to assess their effect on withdrawal response was 5×10-
6 M for group VII.
Group VIII--Gliclazide treatment (50×10-6 M) + Morphine-naloxone response): Basal
naloxone contraction was obtained in the morphine dependent rat ileum. After 10 min resting
periods, the same tissue was subjected to gliclazide treatment (dissolved in 0.9% saline) along
with morphine treatment and the effect of naloxone on such a tissue was then tested. The
concentrations of gliclazide achieved in the bath employed to assess their effect on withdrawal
response was 50×10-6 M for group VIII.
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Electronic Supplementary MaterialJournal: Psychopharmacology
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Figure 3: Raw traces showing effect of various treatments on morphine naloxone induced contraction (during morphine withdrawal) on rat ileum.ACh: acetylcholine (10-6 M); M: Morphine (10-5 M); N: naloxone (10-5 M); I: insulin (10×10-6 M &1×10-6 M); G: gliclazide (5×10-6 M and 50×10-6 M); D1: dose 1; D2: dose 2
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Figure 3
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